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. Author manuscript; available in PMC: 2017 Jun 15.
Published in final edited form as: J Immunol. 2016 May 9;196(12):4977–4986. doi: 10.4049/jimmunol.1502044

Figure 5. miR-182 targets Foxo3a and Maml1 inhibit TNF-α-mediated inflammatory osteoclastogenesis.

Figure 5

(A) BMMs transfected with either control or Maml1 siRNA (80 nM) were stimulated with TNF-α for 6 days. TRAP staining was performed (Left) and the number of TRAP-positive MNCs (>= 3 nuclei per cell) per well relative to the control was calculated. Data in figure represent average ± SEM of four independent experiments and statistical significance was calculated by paired-t-test *P<0.05 (Right). Bar, 200 µm. (B) Quantitative real-time PCR analysis of mRNA expression of Acp5 (encoding TRAP), Ctsk (encoding cathepsin K) and Itgb3 (encoding β3) in BMMs transfected with control or Maml1 siRNA (80nM) and stimulated with or without TNF-α for 3 days. Data are representative of and statistical testing was performed on three independent experiments. *P<0.05. (C) BMMs transfected with either control or Foxo3a siRNA (80 nM) were primed with RANKL (40 ng/mL) overnight followed by TNF-α (40 ng/mL) stimulation for 4 days. TRAP staining was performed (Left) and the number of TRAP-positive MNCs (>= 3 nuclei per cell) per well relative to the control was calculated. Data in figure represent average ± SEM of three independent experiments and statistical significance was calculated by paired-t-test *P<0.05 (Right). Bar, 200 µm. (D) Quantitative real-time PCR analysis of mRNA expression of Acp5 (encoding TRAP), Ctsk (encoding cathepsin K) and Itgb3 (encoding β3) in BMMs transfected with control or Foxo3a siRNA (80nM) and stimulated by RANKL priming and TNF-α for 3 days. Data are representative of and statistical testing was performed on three independent experiments. *P<0.05. (E, F) Toluidine blue-stained dentin resorption pits (left panel) formed by the osteoclasts derived from wild type BMMs transfected with control or Maml1 siRNA (80 nM, E) or Foxo3a siRNA (80 nM, F) in the presence of TNF-α (40 ng/ml) for 10 days without (E) or with RANKL (40 ng/ml) priming for overnight (F). Bar, 200 µm. Relative pit area (% to slice) is shown in the right panel. Data are representative of and statistical analysis was performed on three independent experiments. *P<0.05.